Project Summary The proposal's objective is to identify the mechanisms that support microglia-mediated suppression of dopamine responses. Microglia contribute to normal brain development by supporting neuronal survival and brain tissue clearance from non-functional neurons and synapses. We identified a novel function of microglia that involves the suppression of dopamine-induced behaviors in the adult brain. We found that mice with a pan-brain or striatum-specific ablation of microglia display exaggerated dopamine- induced motor activity and a propensity to seizures. These findings suggest a suppressive effect of microglia on striatal neuron activation. Our preliminary data suggest that this effect could be elicited directly by dopamine-induced microglia activation. We found that 10-15% of microglia in the striatum but not in other brain regions express the dopamine D1 receptor. The expression of the dopamine D1 receptor by a subpopulation of striatal microglia suggests a potential novel negative feedback mechanism where microglia tune neuronal response to dopamine after directly sensing/responding to changes in the neurotransmitter level. Our proposal aims to elucidate the mechanism underlying microglia-mediated suppression of dopamine responses and revolves around the following major questions: Is there a specific subpopulation of striatal microglia that controls neuronal suppression, and if yes, is it based on direct microglia triggering by dopamine? Does the suppressive activity of microglia target a specific subpopulation of striatal neurons? What is the nature of the microglia-produced mediators that impact neuronal responsiveness to dopamine? Our proposal relies heavily on in vivo animal models, and we plan to generate new strains of transgenic mice that enable the manipulation of microglia in the striatum. One of the innovative methodological aspects of the proposal involves state of the art cell type-specific gene expression analysis in different neurons and microglia populations using techniques developed by us that minimize aberrant changes in gene expression caused by cell isolation procedures. In addition to elucidating the specific striatal microglia and neuron subpopulations mediating the suppressive effect, our proposal aims to identify the microglia-produced regulators of neuronal responses to dopamine. We found that the suppressive activity of microglia requires the expression of the bromodomain-containing protein Brd4, which functions as a regulator of gene transcription. Our data suggest that Brd4-bound microglia genes, especially those that are upregulated by dopamine, may encode microglial suppressors of dopamine responses. Identification of these genes is one of the major goals of the proposal, which we plan to achieve by using microglia-specific RNA and chromatin analysis developed by us. Overall, our proposal has the potential to identify novel microglia- based mechanisms that regulate neuronal responses to dopamine.